Fos gene products in CeA- and BNSTov-CRF cells remarkably suggesting that their contribution to stress adaptation response does not require AP1-controlled transcriptional changes.Corticotropin-releasing factor (CRF) immunoreactive (ir) neurons of the paraventricular nucleus of the hypothalamus (PVN) play pivotal role in the coordination of stress response. CRF-producing cells in the central nucleus of amygdala (CeA) and oval division of the bed nucleus of stria terminalis (BNSTov) are also involved in stress adaptation and mood control. Immediate early gene products, subunits of the transcription factor activator protein 1 (AP1) are commonly used as acute (FOS) and/or chronic (FOSB/deltaFOSB) markers for the neuronal activity in stress research. It is well known that the course of aging affects stress adaptation, but little is known about the aging-related stress sensitivity of CRF neurons. To the best of our knowledge, the stress-induced neuronal activity of CRF neurons in the course of aging in acute and chronic stress models was not studied systematically yet. Therefore, the aim of the present study was to quantify the acute restraint stress (ARS) and chronic variable mild stress (CVMS) evoked neuronal activity in CRF cells of the PVN, CeA, and BNSTov using triple-label immunofluorescence throughout the whole lifespan in the rat. We hypothesized that the FOS and FOSB content of CRF cells upon ARS or CVMS decreases with age. Our results showed that the FOS and FOSB response to ARS declined with age in the PVN-CRF cells. BNSTov and CeA CRF cells did not show remarkable stress-induced elevation of these markers neither in ARS, nor in CVMS. Exposure to CVMS resulted in an age-independent significant increase of FOSB/delta FOSB immunosignal in PVN-CRF neurons. Unexpectedly, we detected a remarkable stress-independent FOSB/deltaFOSB signal in CeA- and BNSTov-CRF cells that declined with the course of aging. In summary, PVN-CRF cells show decreasing acute stress sensitivity  with the course of aging, while their (FOSB/deltaFOSB) responsivity to chronic challenge is maintained till senescence. Stress exposure does not affect the occurrence of the examined  Fos proto-oncogene family and contribute to the modulation of gene expression as subunits of the well-known transcription factor activator protein 1 (AP1). Fos is a commonly used neuronal activity marker to study the acute neuronal response that occurs minutes after the stimulus at mRNA level  products. For instance, FOS  and FOSBNA level . At protNA level  and finaNA level .FosB shows increase both in its transcription and translation to FOSB protein upon exposure to stimuli that require neuronal response at the level of gene expression  might be affected by age . In our Crf expression increases upon stress exposure at mRNA level  controls a wide spectrum of physiological functions affecting the stress response  axis. Indeed, PVN NA level  in the rNA level  and chroNA level  results NA level , 2008. Irticoids . After tresponse  immune sresponse , energy response , reproduresponse .Limbic areas, such as nuclei of the extended amygdala, were also shown to contribute to the stress adaptation response  acute and (b) chronic stress reactivity of CRF neurons in the PVN, CeA, and BNSTov was affected by the course of aging in the rat. To test these hypotheses, (a) male rats of eight age groups , CeA [(−2.40 mm) to (−2.92 mm) to the Bregma], and BNST [+0.12 mm to (−0.24 mm) to the Bregma] were manually selected according to the rat brain atlas by Selected sections were rinsed 4 × 10 min in PBS. A heat-induced epitope retrieval was applied at 90°C in citrate buffer (pH = 6.0) for 10 min. Sections were left in the same solution to cool down to room temperature for additional 20 min. Then, sections were washed for 2 × 10 min in PBS and permeabilized with 0.5% Triton X-100 for 60 min . Subsequently, after an incubation for 60 min in 2% normal donkey serum  in PBS, sections were treated with the mixture of polyclonal rabbit CRF antiserum diluted to 1:16,000 , FOS polyclonal guinea pig antiserum diluted to 1:1000 , and polyclonal mouse FOSB antiserum diluted to 1:1500  in NDS for 48 h at 4°C. After 2 × 15 min PBS washes, sections were incubated for 24 h in a cocktail of the following secondary antisera purchased from Jackson: biotinylated donkey anti-rabbit IgG (1:500), Alexa 488-conjugated donkey anti-guinea pig IgG (1:600), Cy3-conjugated donkey anti-mouse IgG diluted to 1:400 in PBS. After repeated washes in PBS, preparations were treated with Cy5 streptavidine  for 3 h. To avoid the influence of the autofluorescence due to aging-related lipofuscin accumulation, an autofluorescence eliminator kit was used according to the supplier’s instructions with slight modifications . Sections after immersion in PBS for 5 min were transferred into 30% and then into 70% ethanol solution for 5 min, respectively. Then, sections were immersed into the autofluorescence eliminator reagent for 2 min, followed by 2 × 1 min treatment with 70% ethanol solution. Finally, sections were rinsed 2 × 15 min in PBS, mounted on gelatin-covered slides, dried on air, and covered with a solution of PBS and glycerol (1:1).The specificity and sensitivity of the FOS antiserum  was tested on our samples. Omission of the primary or secondary antiserum, their replacement with normal non-immune sera abolished the immunosignal in this experiment. Pre-incubation of the working dilution of the antiserum with the respective blocking peptide  prevented the immunolabeling also . The FOSAn experienced neuro-histologist colleague who was unaware of the identity of the preparations digitalized the sections containing the PVN, CeA, and BNST areas by Olympus FluoView 1000 confocal microscope. Sequential scanning in photon count mode was used for the respective fluorophores to detect semi-quantitative fluorescent signal. The confocal aperture was set to 80 μm. The scanning was performed with a 20× objective to obtain images of 1024 × 1024 pixel resolution. The excitation and emission spectra of the respective fluorophores were set according to the built-in settings of the Fluo-View software . 488, 550, and 633 nm laser beams were used to excite Alexa 488 (emission peak for detection: 525 nm), Cy3 (emission peak for detection: 570 nm), and Cy5 (emission peak for detection: 670 nm), respectively.Images of respective channels were stored both individually, and superimposed to evaluate the co-localization of fluorescent signals. Red (Cy3), green (Alexa 488), and white (Cy5) virtual colors were used.The number of the cells was counted in non-edited digital images by a colleague who wasn’t informed about the identity of sections and the purpose of the study. The quantitation of neurons was performed on five digital images of each brain area of interest selected from one series of representative sections. All cells were counted on the entire cross-section surface of the respective nuclei. Cell counts of the five sections were averaged. These numbers represented one animal in the statistical analysis. In order to provide a semi-quantitative analysis of neuronal CRF content, the intensity of fluorescence in the perikarya was measured and corrected for the background signal, yielding the specific signal density  and 15 nl/tube CS-RCS-57 antibody .Data were presented as mean of groups ± standard error of the mean (SEM). The normality of data distribution and the homogeneity of variance were verified by Shapiro–Wilk  and HartIn order to test the potential age dependency in CRF neuronal activity upon acute stress exposure, the FOS and FOSB immunoreactivities were assessed in the PVN, CeA, and BNSTov. Eight age groups of rats were compared with or without ARS exposure.F = 74.39; p < 10–6) and acute stress  = 7.72; p < 10–5) affected the number of CRF-FOS double labeled cells  = 7.68; p < 10–5). Tukey’s post hoc test revealed that 1M, 1.5M, and 12M animals upon acute stress exposure showed significantly elevated CRF-FOS cell counts compared to their respective controls (p < 0.0005). Spearman’s correlation test .Analysis of variance  revealeded cells . Moreoveion test  verifiedF = 89.17; p < 10–6), age  = 2.67; p < 0.05), and their interaction  = 3.03; p < 0.01) on the number of CRF-FOSB double positive cells significant (post hoc test confirmed a significant increase in CRF-FOSB cell counts in ARS groups vs. their age-matched controls in 1M and 1.5M groups (p < 0.0005). Age negatively correlated (p < 0.0005). The ARS-induced PVN-CRF-FOSB cell count positively correlated with CRF-FOS .Analysis of variance  revealednificant . The posrrelated  with theF = 7.35; p < 10–5), age  = 66.81; p < 10–6), and their interactions  = 6.78; p < 10–5) on the number of CRF-FOS-FOSB triple positive cells significant  and 1.5M (p < 0.0005) ARS rats. The Spearman’s correlation analysis (p < 0.001). The analysis of CRF SSD revealed that the CRF content of triple positive cells correlated with age  and with IEG product content , while the CRF content of cells which were immuno-negative for IEG products did not show significant correlation  with the count of IEG product double positive cells. It should be pointed out also that if one activity marker was present in the nucleus of the CRF neurons upon acute stress, the other mostly co-existed also as both CRF-FOSB  and CRF-FOS  strongly correlated with CRF-FOSB-FOS  = 6.04; p < 0.05) and age  = 3.45; p < 0.01) on CRF-FOSB co-existence significant (p < 0.05). This was verified by a weak correlation .In the CeA, ANOVA  revealednificant . AlthougF = 4.13; p < 0.05) and age  and 1.5M ARS animals (p < 0.01). However, the main effect of stress was significant; the post hoc tests did not confirm the ARS-induced elevation of FOSB  = 3.54; p < 0.01) and age  = 30.73; p < 10–5) as well as their interaction  = 4.55; p < 0.001) significantly influenced the CORT level (p < 0.0001) reacted with a significant CORT rise. In the case of 2M and 12M rats, the CORT level showed some increase upon ARS, but the elevation did not reach the statistically significant value according to the post hoc test  supporting the validity of our CVMS protocol. CORT  = 3.02; p < 0.02) exposure and by age  = 13.96; p < 0.001) without an interaction  = 1.74; p = 0.14). Post hoc comparisons revealed that the CVMS exposure caused a significant elevation in 3M rats. In older CVMS exposed animals, the CORT level decreased with age  = 5.86; p < 0.05) and age  = 60.54; p < 10–6) as well as their strong interaction affected the animals’ total bodyweight  = 10.96; p < 10–6). The CVMS-related bodyweight decline was significant when control and CVMS rats were exposed in the 2M and 3M groups .Both CVMS  = 19.66; p < 10–4) and age  = 5.86; p < 0.0005) influenced the relative adrenal weight .The main effect of both stress  = 175.73; p < 10–6). The CMVS per se did not affect relative thymus weight, however, there was a marked effect of stress and age interaction  = 4.00; p < 0.005). 2M control and CMVS animals showed significantly higher thymus weight/bodyweight ratio than all other groups , except 3M controls  = 64.19; p < 10–6). In contrast, age did not influence the FOSB reactivity of CRF neurons (post hoc test confirmed the significant rise of CRF-FOSB cell counts upon CVMS in 3M and 18M groups (p < 0.0005) although a similar trend was observable in all age groups without statistical significance. In order to test if the FOSB affected the CRF content of the cells, the SSD of CRF ir was determined. We found that the CRF content of the cells correlated positively with the FOSB ir  with the magnitude of FOSB signal.The number of cells which contained both CRF and FOSB was strongly affected by CVMS exposure in the PVN  = 3.93; p < 0.005) only .The number of CeA cells co-localizing CRF and FOSB was affected by age (ANOVA: 05) only . The ageF = 5.37; p < 0.001; post hoc test found that the magnitude of CRF-FOSB cell count decreased with age  and the latter with the relative thymus weight  also.The assessment of CRF-FOSB ir in the BSNTov revealed that similar to the CeA, the number of CRF-FOSB double positive neurons was affected by age  content of CRF neurons neither in CeA nor in BNSTov upon CVMS exposure. This is in line with our  and othe stimuli . The sigIn the present study we also detected FOS in the ARS model at 2 h successfully, which is in line with earlier studies . One of First, the visualization of FOS proteins provides information about the activity of the AP-1 complex , but doeTo the best of our knowledge, this is the first study providing a systematic throughout-lifespan description of neuronal activity in the main hypothalamic and forebrain CRF systems in response to ARS and CVMS in the rat. The neuronal activity of the examined CRF cells was found to be a function of age and brain area. CVMS was found to cause an age-independent FOSB/ΔFOSB neuronal activity in PVN-CRF cells. Therefore, studies applying FOS and FOSB as activity markers should be planned with respect to the age and brain region-specific recruitment of these indicators. Further studies are in progress to describe the aging-related alteration of neuronal stress responsivity in other stress-recruited circuits. Getting an overview on the age-dependency of stress-reactive areas may help to understand why do stress-related brain diseases occur more frequently in adolescence and in old age. This knowledge might ultimately help to find new personalized, eventually age-adjusted strategies for prevention and management of stress-related mood-disorders.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Ethics Committee on Animal Research of Pécs University.LK performed the animal experiments, evaluated the results, performed the statistics, prepared the figures, and wrote the draft of the manuscript. NF and BU performed the immunolabeling, digital imaging, and morphometry. GB contributed to the work at the confocal microscope. VC performed and validated the corticosterone measurement. BG designed the experiments, collected the blood samples, performed the perfusion, supervised the tissue preparation, imaging, selected the images containing the areas of interest, helped with figure preparation, and supervised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brain vessel status is a promising biomarker for better prevention and treatment in cerebrovascular disease. However, classic rule-based vessel segmentation algorithms need to be hand-crafted and are insufficiently validated. A specialized deep learning method—the U-net—is a promising alternative. Using labeled data from 66 patients with cerebrovascular disease, the U-net framework was optimized and evaluated with three metrics: Dice coefficient, 95% Hausdorff distance (95HD) and average Hausdorff distance (AVD). The model performance was compared with the traditional segmentation method of graph-cuts. Training and reconstruction was performed using 2D patches. A full and a reduced architecture with less parameters were trained. We performed both quantitative and qualitative analyses. The U-net models yielded high performance for both the full and the reduced architecture: A Dice value of ~0.88, a 95HD of ~47 voxels and an AVD of ~0.4 voxels. The visual analysis revealed excellent performance in large vessels and sufficient performance in small vessels. Pathologies like cortical laminar necrosis and a rete mirabile led to limited segmentation performance in few patients. The U-net outperfomed the traditional graph-cuts method . Our work highly encourages the development of clinically applicable segmentation tools based on deep learning. Future works should focus on improved segmentation of small vessels and methodologies to deal with specific pathologies. Stroke is a world disease with extreme impact on patients and healthcare systems. Approximately 15 million people suffer from an ischemic stroke each year worldwideCurrently, however, vessel imaging is only visually—qualitatively—assessed in the clinical routine. Technical challenges of extracting brain arteries and quantifying their parameters have prevented this information from being applied in the clinical setting. If done at all, segmentations of brain vessels are to date still done predominantly manually or semi-manually and are not quantified. Additionally, (semi-) manual vessel segmentation is very time-consuming and has proven to be fairly inaccurate owing to high interrater-variability making it unfeasible for the clinical setting  images of patients with cerebrovascular disease. We performed a thorough qualitative and quantitative assessment to assess performance with a special focus on performance for pathological cases. Lastly, we compared our results to a traditional standard method of the graph cut approach  or internal carotid artery (ICA)]. The study details have been published previously  with Time-of-Flight (TOF)-angiography from a different scanner and different parameters, and a different angiography modality, i.e. MPRAGE-angiography .For the PEGASUS patients, scans were performed on a clinical 3T whole-body system  using a 12-channel receive radiofrequency (RF) coil  tailored for head imaging.3; matrix size: 312 × 384 × 127; TR/TE = 22 ms/3.86 ms; time of acquisition: 3:50 min, flip angle = 18 degrees.Time-of-Flight (TOF) vessel imaging was performed with the following parameters: voxel size = (0.5 × 0.5 × 0.7) mmFor the additional 7UP test-sets, scans were performed on a clinical 3T whole-body system  and a 12 channel RF receive coil  for TOF-imaging and a 7T whole-body system  with a 90 cm bore magnet , an avanto gradient system  and a 1/24 channel transmit/receive coil  was used for MPRAGE-angiography.The parameters were:3; matrix size: 384 × 384 × 160; TR/TE = 24 ms/3.60 ms; time of acquisition: 5:54 min, flip angle = 18 degrees.TOF imaging: voxel size = (0.6 × 0.6 × 0.6) mm3; matrix size: 384 × 384 × 240; TR/TE = 2,750 ms/1.81 ms; time of acquisition: 5:40 min, flip angle = 25 degrees.MPRAGE imaging: voxel size = (0.7 × 0.7 × 0.7) mmmri_nu_correct.mni tool integrated in freesurfer (website: Freesurfer mri_nu_correct.mni)Brain Extraction Tool (BET) of FSL (website: BET/UserGuide-FslWiki)Nipype wrapper implemented in PythonThe raw PEGASUS study TOF images were denoised using the oracle-based 3D discrete cosine transform filter (ODCT3D) implemented in matlab  and MPRAGE-BET parameters (frac 0.15).regiongrowingmacro module implemented in MeVisLab (website: MeVisLab)For PEGASUS TOF data, ground-truth labels of the brain vessels were generated semi-manually using a standardized pipeline. Pre-labeling of the vessels was performed by a thresholded region-growing algorithm using the regiongrowingmacro framework, followed by the above described thorough manual correction steps. Images were reviewed in the final step by VIM.Additional test-set label data (TOF and MPRAGE imaging) was generated using the U-net model in a first step instead of the For U-net training, both PEGASUS TOF images and ground-truth labels were skull-stripped using the whole-brain masks. The data was split into training, validation, and test-sets with 41, 11, and 14 patients-scans, respectively. For illustration of the extracted data see In order to successfully train our deep neural network we needed to consider two challenges. First, the brain slices, with 312 × 384 voxels, are very large and cannot processed at once due to the limited GPU memory. Second, the distribution of vessels within a slice is largely skewed as only 0.9% of brain voxels are vessels. To solve these problems we extracted 1,000 quadratic patches per patient: 500 random patches with a vessel in the center and 500 random patches without a vessel in the center. The model was trained using 4 different patch sizes  and was later tested for best results against the validation set as part of the optimization process. Due to computational limitation the maximal patch size was set to 96 × 96 voxels. Testing the effect of different patch sizes on the model performance would reveal important information about the relevant spatial scope for a reliable vessel detection. The data was normalized patch-wise using zero-mean and unit-variance normalization.The U-net CNN model architecture was adapted from the framework presented by Ronneberger et al. . The U-nA variation of the U-net model architecture was applied, where the number of channels in each layer was consistently reduced to half. For simplicity, the additional architecture is therefore termed throughout this work as “half U-net.”The network is fed with 2D image patches and returns the 2D segmentation probability map for each given patch.The skull-stripped denoised TOF input images and the corresponding ground-truth segmentation maps were used to train the U-net using the Keras implementation of Adam optimizer  = 1/(1 + exp(a(x))) Where a(x) denotes the activation in the final feature channel at the voxel position x ∈ Ω with Ω ∈ Z2 and p(x) is the approximated probability of a voxel x to be a vessel. The Dice coefficient D between two binary volumes is officially defined as TP is the number of true-positive voxels, FP is the number of false positive voxels and FN is the number of false negative voxels. Using the following derivation:In the model, the energy function is computed by a pixel-wise sigmoid over the final feature map combined with the Dice coefficient loss function. The sigmoid is defined as The Dice coefficient can be written as:px ∈ P:Ω → {0, 1} is the predicted binary segmentation volume, gx ∈ G:Ω → {0, 1} is the ground-truth binary volume and s = 1 is an additive smoothing factor . The Dice coefficient then penalizes at each position j the deviation of px from the true label gx using the differentiated gradient:Where , false positives (FP), and false negatives (FN) were assigned different voxel values (True negatives (TN) remained labeled with 0). The images were then visualized by overlaying these new masks with the original scans using ITK-Snap (website: ITK-SNAP)Large vessels, overall impression ; Small vessels, overall impression ; Large vessels, errors (FP or FN dominating); Small vessels, errors (FP or FN dominating); Pathology detected (yes/no); Other tissues type segmentation errors (yes/no).All 6 models were applied on the additional 10 7UP patients with different TOF parameters as well as 10 patients with a different angiography modality (7T MPRAGE angiography). Segmentation quality was compared vs. the semimanual gold-standard labels as described above quantitatively with the EvaluateSegmentation framework using as metrics Dice, 95HD and AVD.All trainings ran on a GPU workstation with 16 GB RAM, Intel(R) Xeon(R) CPU E3-1231 v3 @ 3.40GHz and a NVidia TITAN X  GPU with 12 GB VRAM.The U-net model was trained on 81,000 extracted and augmented patches of 41 patients, validated using 11 full patient volumes and assessed for performance using the test-set of 14 full patient volumes. The U-net architecture resulted in 31,377,793 parameters, while the half U-net resulted in 7,846,081 parameters. For the U-net and half U-net, respectively, model training ran for ~100 and 50 min, while segmentation of a previously unseen volume took about 20 and 10 s. The optimal patch-size was identified as 96 × 96 voxels for both suggested architectures. The model parametrization can be found in The U-net model yielded high performance in terms of the three measures that were comparable for both the full and the half architecture: The models optimized for the Dice coefficient had a Dice value of 0.89. The 95HD value was 47 voxels and the AVD models yielded results around 0.35 voxels. The detailed performance assessment of the finalized models is presented in The visual analysis of the three full models showed that consistently in all 14 patients the large vessels were segmented excellently. Only very few false positive voxels in the border zones of the vessels were present see . Small vA comparison of the three models showed comparable performance and consistent artifacts as described above. There was a tendency, however, for 95HD and AVG models to have less false positively labeled meningeal and venous structures than the Dice-optimized model. The visual comparison of the full and the half architecture showed comparable performance in the large vessels. We saw a tendency for slightly worse performance in smaller vessels in the half architectures. Vessel pathologies (stenosis/occlusion) were depicted in all patients and all models.Graph cut results showed inferior performance to the U-net models with the following results (patch/whole slice): Dice 0.76/0.76, 95 HD 58.8/59.2, and AVD 1.97/1.97 . Detailed results can be found in We present in the current work a U-net deep learning framework for fully automated brain arterial vessel segmentation from TOF-images of patients with cerebrovascular disease. Our framework demonstrated a very high quantitative performance based on three validation metrics. A lighter architecture—half U-net—achieved comparable quantitative performance. Visual inspection showed excellent performance in large vessels and sufficient to good performance in small vessels as well as comparable performance between the full architecture and the half-net. Special cerebrovascular pathologies presented challenges for the network and need to be addressed in the future.Applying a modified U-net framework as suggested by Ronneberger et al. , we achiNext to a quantitative assessment, an experienced medical professional also visually assessed the quality of the segmentations. We found that also in the visual analysis the performance of the networks was very high. However, while we saw excellent performance for large vessels, the performance in smaller vessels was less pronounced. While future networks should be improved regarding small vessel segmentation, the clinically most relevant vessels are the large vessels. Thus, we present evidence that already a relatively simple U-net architecture shows clinically highly relevant performance. Even higher performance can be expected using newer segmentation architectures, e.g., the MS-net  for data augmentation, e.g., by Antoniou et al.  few training examples are available . Thus, our results are more representative of the clinical challenges than the results of works using the data of healthy patients. And indeed, we found that special pathological cases like cortical laminar necrosis and a rete mirabile were challenging for the network and need to be focused in future works. In summary, our work serves as the starting point to develop new pathology-tailored models which are applicable in the clinical setting. In their training, random patch extraction should be avoided, and patch selection should be focused on the special cases identified in our study. Second, we labeled 66 patients, which is—in medical imaging—a large number of patients. This number is roughly double to triple of the number of the datasets used by Ronneberger et al. in their original U-net paper  Since clinical on-site postprocessing is tied to the scanner-vendor and software, segmentation products tuned for vendor-specific sequences and parameter-ranges are possible and lack of generalization is unproblematic. (2) For development of a vendor-independent pipelines, clinical segmentation algorithms need to cover a much broader range of image variability. Here, a large number of varied datasets needs to be used for training of single models or model zoos in the future. Second, we reconstructed the images on the patch level and did not perform an algorithm-based optimization of the whole reconstructed vessel tree. Here, future works can explore for example recurrent neural networks, especially architectures with long short-term memory (LSTM) layers. Applying these techniques, an increase of small vessel segmentation performance might be possible. Third, also, the patch size was limited due to hardware constraints potentially reducing the performance of the network where more context is needed. Fourth, we performed an exploratory qualitative visual analysis by one medical expert. Future clinical assessments of different models should include a systematic quantitative rating by multiple medical expert readers, which exceeded the scope of the present work.In conclusion, a U-net deep learning framework yielded high performance for vessel segmentation in patients with cerebrovascular disease. Future works should focus on improved segmentation of small vessels and removal of artifacts resulting from specific cerebrovascular pathologies.ML, TK, JS, JDK, KH, DF, and VM: concept and design; VM and JS: acquisition of data; ML, JR, AT, JDK, KH, and VM: model design; ML, JR, OA, EA, DF, and VM: data analysis; ML, JR, AT, TK, JS, JDK, KH, DF, and VM: data interpretation; ML, JR, OA, AT, EA, TK, JS, JDK, KH, DF, and VM: manuscript drafting and approval.While not related to this work, JS reports the following board memberships, consultancies, and/or payments for lectures including service on speaker's bureaus: Boehringer-Ingelheim, Sanofi, Bayer, Pfizer, and Maquet. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The central sensitization inventory (CSI) comprises 25 items and is commonly used to measure somatic and emotional symptoms related to central sensitization symptoms. CSI was developed as an easy-to-administer screening instrument for patients at high risk of developing central sensitization in whom it was essential to quickly evaluate the condition. The purpose of the present study was to develop a short form of CSI and evaluate its psychometric properties using a contemporary approach called Rasch analysis.A total of 505 patients with musculoskeletal disorders were recruited in this study. The CSI, pain intensity, pain interference, and the health-related quality of life (QOL) were evaluated for each participant. The original CSI items were consecutively analyzed using the Rasch model. Successive Rasch analyses were performed until a final set of items satisfied the model fit requirements. We also analyzed the psychometric properties of the original and short forms of CSI.Four consecutive Rasch analyses identified the removable items. Finally, the shortest questionnaire obtained that maintained the correct psychometric properties based on the Rasch model contained only 9 items (CSI-9). Rasch analysis showed that the CSI-9 had acceptable internal consistency, exhibited unidimensionality, had no notable differential item functioning, and was functional on the category rating scale.The nine-item short form of CSI has acceptable psychometric properties and is suitable for use for patients with musculoskeletal pain. Thus, the CSI-9 can be used as a brief instrument to evaluate central sensitization. The International Association for the Study of Pain defines central sensitization (CS) as an increased responsiveness of nociceptive neurons in the central nervous system to normal or subthreshold afferent input . SeveralThe CSI total scores have been shown to distinguish between patients with chronic pain and control subjects , 10. A hCSI has been translated into many languages and has been validated by a variety of practitioners, 16–21. The Rasch model , which iIndividuals aged between 20 and 80 years were consecutively recruited and screened by orthopedists to receive physical therapy from an orthopedic clinic. Those with acute (pain that lasts less than 3 months) or chronic (pain that lasts for at least 3 month) musculoskeletal pain disorders  were included. All participants underwent X-ray examination before receiving physical therapy. At the screening stage by orthopedists, participants with fracture, sprain, cancer, multiple sclerosis, brain or spinal cord injury, history of stroke, or history of psychiatric disease  as diagnosed by a psychiatrist were excluded.All evaluations were performed before physical therapy. Of the initial sample of 510 participants, 5 participants were excluded because they did not complete all the items in the questionnaires. A total of 505 individuals were included; all these patients were Japanese. Their characteristics are summarized in Ethical approval was obtained from the Institutional Ethics Committee of Konan Woman's University. Written informed consent was obtained from all the participants before the study, and the study was conducted in accordance with the tenets of the Declaration of Helsinki.All participants were assessed for demographic data , pain duration, CSI, health-related quality of life (QOL), pain intensity, and pain interference. CSI-J consists of Parts A and B. Part A consists of a 25-item self-report questionnaire designed to assess health-related symptoms that are common to CSS. Each item is rated on a 5-point Likert-type scale, with total scores ranging from 0 to 100. Part B (which is not scored) asks the participants whether one or more specific disorders, including seven separate CSSs, have been diagnosed previously .Health-related QOL was measured using the EuroQol 5-dimension (EQ-5D) instrument . EQ-5D wPain intensity and interference were measured using the Brief Pain Inventory (BPI) , 43. BPIThe original CSI was consecutively analyzed using the Rasch model by Winsteps software . Chi-square fit statistics were used to determine how well each CSI item contributed to total CSI scores . The mosWe investigated the psychometric properties of the original and shortened questionnaires, and compared the following components:We assessed category order to ensure the Likert scale functioned as expected. The CSI has five response categories . Category probability curves, and average measure and category fit statistics (infit and outfit) were used to explore rating scale functioning. Fit statistics are recommended to be between 0.6 and 1.4, 50. MorScale-to-sample targeting was made by comparing person locations from the sample with item locations of the scale. Their means should be close to 0 logits. In addition, we investigated targeting by visualizing the person-item map and comparing the means of the items and person measures. This was analyzed by plotting the person-item location threshold distribution graph with distributions of persons on the top half of the graph and item thresholds at the bottom half of the graph. Good targeting means that the distributions of persons and distributions of item thresholds match visually.Winsteps provides a person reliability index and Cronbach’s alpha as indicators of internal consistency  and bothWe intended to summate the CSI to provide an overall measure of CS-related symptomology. Individual items should share this unidimensional construct yet be sufficiently different to warrant their inclusion. Assessment of fit evaluates unidimensionality by identifying items that function unexpectedly. The principal component analysis of residuals (PCA) identifies unexpected clusters of items suggestive of a secondary dimension that could threaten measurement of the primary dimension. The PCA residual correlation matrix was visually inspected to identify clusters of items that would be suggestive of a second dimension. An eigenvalue greater than 2.0 for the PCA of residuals suggests a second dimension . ResponsThe dimensionality of both versions was also explored using exploratory structural equation modeling (ESEM) with geomin oblique rotation in Mplus 8. We tested various models: from a one-factor model to a five-factor model in the CFI-25 and from a one-factor model to a two-factor model in the CFI-9. A minimum cutoff of 0.95 for Comparative Fit Index (CFI), a maximum cutoff of .08 for Root Mean Square Error of Approximation (RMSEA), and a maximum cutoff of 0.08 for Standardized Root Mean Square Residual (SRMR) were considered as indicative of acceptable fit –58. ModeParticipants with outfit residuals greater than 1.5 logits were examined to determine the reason for poor fit. They were compared with those who fit the model using Fisher’s exact test  of signiItems should function in a similar manner for all people of similar levels of agreeability. We assessed for DIF across 5 subgroups: gender, age , pain intensity from BPI , pain duration , and pain interference from BPI . DIF was tested using a Mantel-Haenszel chi-square test with significance level set at p = 0.01 for each item. Item bias was explored if an item yielded a significant difference of greater than 0.5 logits between subgroups [3,1 values 0–0.40 were considered to indicate fair reliability, 0.41–0.60 moderate reliability, 0.61–0.80 substantial reliability, and 0.81–1.00 almost perfect reliability [CSI reliability was assessed using scores obtained from a second round of the questionnaire administered to participants within 2 weeks of the first questionnaire completion. An intraclass correlation coefficient (ICC) 2-way mixed model with absolute agreement was used to determine measurement reliability. ICCCorrelation analysis was evaluated using the Statistical Package for Social Sciences Version 25 . Data distribution was tested for homoscedasticity using the Kolmogorov-Smirnov test. A series of correlations was performed examining the relationships between the original and the short form of CSI total score and pain intensity, interference, and QOL. These correlations were investigated with Spearman’s correlation coefficient.3,1 and the 95% confidence interval for agreement. ICC3,1 values 0–0.40 were considered to indicate fair reliability, 0.41–0.60 moderate reliability, 0.61–0.80 substantial reliability, and 0.81–1.00 almost perfect reliability [In addition, the correlation between the original and the short form of CSI total score was investigated by calculating the ICCFive severity levels of the original CSI  were recommended to evaluate symptom severity or assess clinical changes in response to treatment . To deteThe differences in age, pain duration, pain intensity, pain interference, and health-related QOL were also compared with the shortened CSI severity level group using ANOVA and Tukey post-hoc analysis. Gender was compared with the shortened CSI severity level group using the chi-squared test. We adjusted alpha to 0.008 because we undertook six separate analyses.Four consecutive Rasch analyses identified the items that could be removed from the questionnaire . FinallyThe sample was not well targeted by both versions of CSI. The average person endorsability of CSI-25 and CSI-9 (mean ± SD logits) were -1.42 ± 0.91 and -1.09 ± 1.11, respectively. The item endorsability averages of CSI-25 and CSI-9 (mean ± SD logits) were 0 ± 0.72 and 0 ± 0.79, respectively. Visually, the shifting of person agreeability of CSI-25 and CSI-9 to the left when compared with item endorsability indicated that participants with low levels of central sensitization were not well targeted by the scale .In CSI-25 and CSI-9, 4 (0.7%) and 7 participants (1.3%), respectively, scored zero for all the items. None of the participants for CSI-25 and CSI-9 scored full points on all items.The average agreeability measures of the respondents advanced as expected across the rating scale categories in both CSI-25 and CSI-9, and there was neither excessive positive nor negative fit statistics in CSI-9, suggesting the category structure is adequate, but, in CSI-25, there was no excessive outfit statistics .Category 4 in CSI-25 was underutilized, which suggests the respondents experienced difficulty differentiating “often” from “always” . Each caPCA of the residuals indicated that the unexplained variance of the first contrast of CSI-25 and CSI-9 was 2.2 and 1.6 eigenvalue units, respectively, indicating multidimensionality of CSI-25 and unidimensionality of CSI-9. The tested five-factor ESEM models in CSI-25 and two-factor model in the CFI-9 were the most fitted to the data .The person reliability of CSI-25 and CSI-9 was 0.90 and 0.77, respectively. Cronbach’s alpha of CSI-25 and CSI-9 were 0.89 and 0.80, respectively. McDonald’s Omega of CSI-25 and CSI-9 were also 0.89 and 0.80, respectively. These results suggest that both versions of CSI have good internal consistency.Analysis of person fit of CSI-25 and CSI-9 identified 73 participants (14.4%) and 70 participants (13.8%) with excessive positive outfit (>1.5 logits), respectively. In CSI-25, the CSI-25 score of participants with misfit were significantly higher than those without misfit .There was differential item functioning for age  in CSI-25, but there was no differential item functioning for age, gender, pain intensity, pain interference or pain duration in CSI-9.3,1 of 0.86  and 0.79 , respectively.Of the participants who reported no change (the difference between first and second round of pain intensity during movement is under 10 mm) in pain intensity during the past 2 weeks (n = 100), in CSI-25 and CSI-9, there was excellent agreement between test and retest total scores, with an ICC3,1 of the CSI-25 and CSI-9 was 0.69 , suggesting substantial agreement.The CSI-9 was significantly correlated with CSI-25  . The CSIOf the 505 participants, 372 (73.6%) reported 0, 96 (19.0%) reported 1, 24 (4.7%) reported 2, and 13 (2.5%) reported ≥ 3 CSS diagnoses. The CSI-9 mean score was 9.9 ± 5.5 in the no CSS group, 13.0 ± 5.0 in the 1 CSS group, 13.7 ± 6.5 in the 2 CSS group, and 20.5 ± 5.7 in the 3 or more CSS group. The CSI score in patients with no CSS was significantly lower than in those with 1, 2, and 3 or more CSS. There was no significant difference in CSI-9 total score between the 1 and 2 CSS groups.ANOVA identified that there was consistent worsening in pain intensity, pain interference, and health-related QOL with respect to the CSI-9 severity levels . The numThe current study aimed to develop a short form of CSI and evaluate its psychometric properties. This is the first study to develop and validate a shortened CSI. The shortened CSI was obtained by reduction of the long and well-validated questionnaires by removing selected items using Rasch analysis. Rasch analysis has been proposed as an objective and optimal method to construct measurement scales and has recently been used to reduce the number of items in other questionnaires. In the present study, CSI-9 had acceptable internal consistency, unidimensional attributes, no notable DIF, test–retest reliability, and was functional on the category rating scale. Overall, CSI-9 showed adequate psychometric properties for use in evaluating health symptoms related to CSS in patients with musculoskeletal pain disorders. Therefore, CSI-9 might be useful in treatment planning when a shortened version of a questionnaire is needed, such as in a time-limited situation or for a specific population.As result of Rasch analysis, 9 items were selected. The face validity of the CSI-9 items is very good. For instance, the items correspond very well with the current criteria for fibromyalgia, using the Widespread Pain Index  and Symp-1.42 logits and -1.09 logits, respectively. Both versions covered average and high levels of somatic and emotional symptoms related to central CSS and are therefore best suited for use with people who have health symptoms related to CSS.The average person endorsability of CSI-25 and CSI-9 were Rating categories complied with the set criteria for category functioning and step measures endorsed monotonically from easy to hard across category responses, supporting proper category order in CSI-9.Although PCA analysis of the residuals of CSI-25 suggests the presence of a second dimension , CSI-9 has unidimensional attributes . This result supports the notion that CSI-9 is appropriate with respect to Rasch analysis. Therefore, a meaningful comparison of CS across patients should be conducted using CSI-9.The internal consistency of CSI-9 was noteworthy  and aligned well with the original English version  [CSI-9 adequately comprises all five components of CSS  in the Japanese version . Therefo3,1 of the CSI-25 and CSI-9 was 0.69, suggesting substantial agreement, thereby making CSI-9 a promising measure.For a satisfactory short form, the reduction in the correlation scores of the short form should not drop by more than 0.10 from the original version . Our resIn the present study, patients were divided into subgroups according to the number of CSS diagnosis. The CSI-9 total score in patients with no CSS was lower than that in patients with 1, 2, and 3 or more CSS, but there was no difference in the CSI-9 total score between participants with 1 and 2 CSS. Therefore, we determined three levels of severity in CSI-9, which included subclinical (0–9), mild (10–19), and moderate/severe (20–36). The study results suggest that a strong relationship exists between the CSI severity groups and pain intensity, disability, and health-related QOL. The lowest CSI severity group reported lower pain intensity, disability, and health-related QOL, whereas the highest CSI severity group reported higher pain intensity, disability, and health-related QOL. These results suggest that the CSI severity groups in CSI-9 have high concurrent and construct validity. Therefore, clinically relevant symptom severity levels provide more useful information to healthcare providers in assessing CS/CSS-related symptoms.Our results are not intended to suggest abandoning CSI-25. CSI-25  has been psychometrically validated in many languages, and its dimensionality and reliability have been established, suggesting that CSI could detect comprehensive traits of CS. The CSI-9 would be a valid and reliable option if researchers or clinicians want to use an abbreviated measure of CSS-related symptoms, such as screening to rule out a disorder or epidemiological surveys.There were several limitations to our study. First, our study included individuals between the age of 20 and 80 years. Therefore, it is possible that age, sex, pain intensity, and pain duration influenced the results of psychometric properties. However, a notable DIF was not found for any of the items in CSI-9, suggesting that there is no effect of participant age, sex, or pain intensity and duration. This finding indicates that these factors might not affect the model. Second, this study included a convenience sample of participants with musculoskeletal disorders who were recruited from a single clinic. Future studies, in which data is compared across multiple centers, are warranted to verify the findings of this study. Third, this study utilized a questionnaire that was psychometrically validated in a Japanese sample and therefore validation of the other language version of the short form of CSI is needed. Fourth, the short form of CSI was extracted from one administration of the full CSI. Further research to investigate the specific CSI short form might be required for more definitive conclusions. Fifth, CSS diagnoses were established from the same self-reported instrument as the CSI scores. This procedure is limited as a validation, similar to internal measure based on two different procedures . Absence of a non-self-reported type and number of clinical diagnosis of CSS is needed in the further study.A nine-item short form of CSI has acceptable psychometric properties and is suitable for use for patients with musculoskeletal pain. Thus, CSI-9 can be used as a brief instrument to evaluate CS.S1 Table(DOCX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


Porphyra yezoensis, separated, and purified using column chromatography with SA-2 cation exchange resin. The effects of MAAs on the activity of endogenous antioxidant enzymes, namely total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) were analyzed in mouse skin tissue. Pathological changes of skin tissue caused by ultraviolet radiation and the arrangement of collagen were observed by Hematoxylin-Eosin (HE) staining and scanning electron microscopy (SEM). Interleukin 1β (IL-1β), IL-6, and IL-10 were detected using the quantitative real-time reverse transcription-polymerase chain reaction (qPCR) and Enzyme Linked Immunosorbent Assay (ELISA). The concentration and expression of these proinflammatory cytokines was associated with the presence of nuclear factor (NF)-κB. The results show that MAA compounds from Porphyra yezoensis could suppress UV-induced photoaging of skin by inhibiting the reduction of endogenous antioxidant enzymes. Compared to the control group, the concentrations of SOD, GSH-Px, and CAT increased significantly in skin tissue homogenate following the external administration of MAAs , while the content of MDA decreased significantly (p < 0.05). Meanwhile, the administration of MAAs was associated with down-regulations in the concentration and mRNA expression of NF-κB, IL-1β, IL-6, and IL-10. The results suggest that MAAs could protect skin from photodamage by increasing antioxidant enzyme activities and inhibiting inflammation. The objective of this research was to extract and prepare mycosporine-like amino acids (MAAs) and investigate the mechanism by which they act against UV-induced skin photoaging in Institute of Cancer Research (ICR ) mice. MAAs such as porphyra-334 and shinorine were extracted from  Skin photoaging refers to cutaneous damage caused by exposure to ultraviolet radiation that leads to skin changes, such as erythema, cornification, melanin sedimentation, and skin aging . When UlWhen the presence of excess ROS results in oxidative stress, the activity of endogenous antioxidant enzymes also decrease. Consequently, the endogenous antioxidant enzyme system, including T-SOD, GSH-Px, and catalase, have significantly lower effects on skin tissue and the level of malondialdehyde (MDA) is significantly higher than under redox balance. This can lead to further UV radiation-induced oxidative damage to skin tissue and photoaging.In UV-induced photoaged skin, ROS trigger signaling transduction and activate nuclear factor-kappa B (NF-κB), a mediator of inflammation . The infMycosporine-like amino acid compounds have been reported to have multiple types of activity, such as ultraviolet light absorption and antioxidant and anti-photoaging activity, as a result of their phenolic hydroxyl structure, which can effectively dispel reactive oxygen species (ROS) . MycospoPrevious studies confirmed that MAA compounds could guard against UV irradiation photoaging by regulating the NF-κB and MAPK signaling pathway and suppressing the expression of matrix metalloproteinases (MMPs) . Based oPorphyra yezoensis using HPLC-UV-MS analysis. The compounds extracted from Porphyra yezoensis were characterized by UV-visible spectroscopy at 200–400 nm, which revealed the maximum UV absorption peak at 334 nm, as shown in m/z) values of 333.1 and 347.1, respectively, as shown in m/z, the sample components were determined to be shinorine and porphyra-334 [Porphyra yezoensis each component were: porphyra-334 = 0.876%, shinorine = 1.039%.Two kinds of major MAA compounds were identified from hyra-334 , the molIn vitro cytotoxicity tests using the MTT assay were carried out to assess the fibroblast cell toxicity of MAA compounds, as shown in HE staining and scanning electron microscopy of Institute of Cancer Research (ICR ) mice skin histological tissue was used to analyze the photoaging damage of mice in each group. The degree of injury to skin keratinocyte cells and collagen fiber is shown in However, vitamin C, which is commonly used in control groups of mice and is an effective antioxidant as assessed by pathology observations, was associated with a tendency for tissue damage migration . SimultaPorphyra yezoensis could effectively relieve skin photoaging damage. This is in agreement with reports that an MAA compound could act effectively against UV damage [The experimental results indicate that the MAA compound extracted from V damage ,17.p < 0.01), and a significantly higher level of MDA (p < 0.01). In contrast, MAA treatment groups were significantly and dose-dependently protected from T-SOD, GSH-Px, and CAT activity  and decreased levels of MDA (p < 0.05).Ultraviolet radiation may cause oxidative damage to skin tissue and cell DNA as a result of the oxidative stress response due to the accumulation of ROS  as reported . The actOf the three MAA treatment groups, the MAAs-H group had the greatest increase in endogenous antioxidant enzyme activity with increases in T-SOD, GSH-Px, and CAT activity in the skin tissue of 37.93%, 34.48%, and 22.22%, respectively compared with irradiated controls (MC). The content of MDA also decreased by 57.14%, as shown in p < 0.01); however, there were no significant differences in the level of IL-10 compared with the NC group (p > 0.05), which may suggest that this factor is not associated with skin photoaging. This illustrates that ultraviolet irradiation could cause a significant rise in the level of pro-inflammatory cytokines. Compared with the MC group, the MAA treatment groups had significantly reduced levels of NF-κB, IL-1β, and IL-6 (p < 0.05 to 0.01), and these effects were dose-dependent. The levels of pro-inflammatory cytokines in mice skin tissue homogenate were measured by using an ELISA assay. As shown in p < 0.05 to 0.01), as shown in Of the three groups of MAA treatment, the MAAs-H showed the greatest inhibition of ultraviolet irradiation-induced pro-inflammatory cytokines, with reductions in NF-κB, IL-1β, and IL-6 activity of 20.9%, 28.33%, and 40.85%, respectively (p < 0.01), by 47.37%, 30.01%, and 34.54%, respectively. Compared with the MC group, the MAA treatment groups had significantly reduced mRNA expression of NF-κB, IL-1β, and IL-6 (p < 0.05 to 0.01), an effect that was shown to be dose-dependent. NF-κB plays an important role in the expression of inflammatory cytokines, which is related to skin photoaging ,19. The p < 0.05 to 0.01), as shown in In this study, of the three MAA dosages, the MAAs-H group better inhibited the expression of NF-κB, IL-1β, and IL-6 mRNA with decreases in expression of 21.05%, 25.10%, and 42.13%, respectively , which accumulate in the intracellular environment and disturb the redox balance. The resulting oxidative stress in skin tissue can lead to inflammation responses, cell death, and skin histopathological lesions, including cancer . SimultaThe ROS-mediated oxidative stress reaction causes an inflammatory response and suppresses the immune system during skin photoaging, during which the content and expression of proinflammatory cytokines continue to rise at different levels . This exOn the other hand, the NF-κB signaling pathway plays an important role in the regulation of the mRNA and the protein expression of inflammatory cytokines . We founPorphyra yezoensis and then separated and structurally confirmed. The commercial kits used to determine the contents of T-SOD, MDA, GSH-Px, and CAT were purchased from Jiancheng Inst . The commercial kits used to determine the contents of NF-κB, IL-1β, IL-6, and IL-10 were purchased from R&D Inst. . The primers targeting mRNAs, including NF-κB, IL-1β, IL-6, and IL-10 were designed and synthesized by Shanghai Biotechnology Co., Ltd., Shanghai, China. Other chemicals and reagents used for this experiment were of analytical grade and are commercially available.MAAs were obtained from Male ICR mice (4 to 6 weeks) were purchased from the Institute of Zoology, Academia Sinica Beijing . All animal procedures for this research conformed to the rules of international moral principles and the Guidelines for the Care and Use of Laboratory Animals . Care anm/z for MS and 60 to 360 m/z for MS/MS. Then the samples were prepared, identified, and stored at –20 ℃ until use.MAA compounds from Porphyra yezoensis were extracted by 15% ethanol aqueous solution. with ultrasonic for 2 hours and purified using SA-2 cation exchange resin, which were applied to analyze further the anti-ultraviolet radiation mechanism. The major bioactive components were analyzed using HPLC-UV-MS and concentration were calculated based on the molar absorptivity of prophyra-334 and shionrine, as previously described ,26. MAA porphyra yezoensis as part of 4.2 by MTT assay . The cultured cells that had been incubated in a 96-well plate  at 100 μL per well were maintained at 37 ℃ in a humidified 5% CO2 incubator for 24 h, and then 10 μL was added to the test substances for model groups . Dulbecco's modified eagle medium (DMEM) was only added to the control group for 12 h. Fifty microliters of MTT reagent (dilution of 1:5) was then added to L929 cells directly for 4 h at 37 ℃. The supernatant was removed, followed by the addition of DMSO (150 μL per well) to dissolve the formazan completely. The plate was then shaken, and the absorbance values of the cells were measured at 570 nm.L929 Fibroblast cells  were used as the cell model to measure the toxicity of MAA compounds extracted from The experiment was performed after all animals had acclimatized for 1 week. They were treated with 6% sodium sulphide-ethanol solution for depilatory unhairing of the back over an area of 2 × 3 cm. It should be noted that the NC group was only treated with unhairing; the MC group received local administration of distilled water (120 μL/d) per mouse on the mouse back skin; the MP group received treatment with vitamin C solution ; and the MAAs-L, MAAs-M, and MAAs-H groups were treated with MAAs solutions from part of 4.2 of 120 μL/d per mouse at respective concentrations of 5, 10, and 20 mg/mL. The solutions were absorbed for 30 min percutaneously, and then all groups were placed under specific conditions involving a combination of ultraviolet irradiations. The UV-induced pathological damage in skin tissues was observed daily after the irradiation experiment. 2 and UVB 18.07 mw/cm2, which total radiation dose were radiation intensities and multiply by the time . In the experiment, all mouse groups received identical doses of ultraviolet light as the distance between the model mice and the UV tubes was adjusted every five minutes. For this modeling process, the damage accumulation method was used to track the appropriate UV irradiation dosing as follows: 0.5 MED  for the first week, and then the UV irradiation intensity was increased per week up to 4 MED. Ultimately, all groups of mice received radiation dosages as follows: UVA: 20.81 J/cm2, UVB: 0.47 J/cm2. The irradiation experiment lasted for about one month.The objective of this experiment was to establish an animal model that, using complex ultraviolet, could be used to research the mechanism by which MAAs regulate tissue antioxidant enzyme levels and the expression of inflammatory factors, thereby protecting against UV irradiation-induced photoaging. The following methods were used for the irradiation of unhaired mouse skin, as in our previous report : One 40 All groups of mice were sacrificed after the last UV irradiation experiment, and dorsal skins were collected so that the level of antioxidant enzymes and inhibition of inflammation in the skin tissue could be further analyzed.Mouse dorsal skin samples were selected from different groups and sliced into one-quarter-inch strips and immersed in 10% buffered formalin for 72 h for tissue fixation. Afterwards, skin tissue was embedded with paraffin wax using the routine method, and then it was cut into slices. The pathological changes of skin tissue were observed with an optical light microscope after HE staining at 40× magnification.In other skin samples, all tissue materials were fixed with 2.5% glutaraldehyde for 7 days and used to analyze the extent of damage done to the local skin tissue and collagen fibers with a scanning electron microscope (SEM) . In addiIn accordance with the antioxidant activity assay directions, 10% skin tissue homogenate was prepared with phosphate buffered saline (1:9 w/v) and stored at −20 °C. The total protein content was measured using protein quantitative kit, and then the SOD, MDA, GSH-Px, and CAT concentrations in the mice skin tissue homogenate were measured and calculated using a reagent kit .The protein expressions of NF-κB, IL-1β, IL-6, and IL-10 in the skin homogenates were measured and calculated using ELISA kit  assays, which were based on the total protein contents of skin tissue homogenates, as previously described ,15. A sample of every skin tissue was ground to a fine powder and the tissue total RNA was extracted using Trizol reagent . Then, the mRNA expressions of NF-κB, IL-1β, IL-6, and IL-10 was determined and analyzed following the instructions of the manufacturer . AgaroseΔΔCt) method following Livak [Using reverse transcription PCR, the RNA extracted from skin tissue was reverse-transcribed to cDNA with M-MLV and random primers . A 25 μL reaction mixture containing SYBR Green Master Mix  and designed specific primer pairs, the prime sequences of which are listed in ng Livak . n = 10). Statistically significance was defined as p < 0.05.All experimental data were analyzed by analysis of variance (ANOVA) with SPSS software . All statistical data are presented as the mean ± standard deviation (Porphyra yezoensis protect against UV irradiation-induced photoaging by adjusting the activities of endogenous antioxidant enzymes and inhibiting inflammation. The results indicate that MAAs can effectively alleviate skin photo-oxidative damage, protect skin tissue, resist UV aging, and sustain the integrity of the structure of collagen fibers and cells. The MAA-related mechanisms of anti-photoaging were shown to be associated with the inhibition of endogenous antioxidant enzyme down-regulation and the suppression of inflammatory cytokine up-regulation and mRNA expression through regulation of the NF-κB signaling pathway. As a result, MAA compounds have the potential to be used as active substances in anti-photoaging skin treatments and inflammation reactions.This research objective of this study was to explore how MAAs extracted from


Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. Radiotherapy, with or without surgery, represents the major approach to curative treatment. However, not all tumors are equally sensitive to irradiation. It is therefore of interest to apply newer system biology approaches  in squamous cancer cells with different radiosensitivities in order to provide new insights on the mechanisms of radiation response. In this study, two cultured HNSCC cell lines from the same donor, UM-SCC-74A and UM-SCC-74B, were first genotyped using Short Tandem Repeat (STR), and assessed for radiation response by the means of clonogenic survival and growth inhibition assays. Thereafter, cells were cultured, irradiated and collected for subsequent metabolic profiling analyses using liquid chromatography-mass spectrometry (LC-MS). STR verified the similarity of UM-SCC-74A and UM-SCC-74B cells, and three independent assays proved UM-SCC-74B to be clearly more radioresistant than UM-SCC-74A. The LC-MS metabolic profiling demonstrated significant differences in the intracellular metabolome of the two cell lines before irradiation, as well as significant alterations after irradiation. The most important differences between the two cell lines before irradiation were connected to nicotinic acid and nicotinamide metabolism and purine metabolism. In the more radiosensitive UM-SCC-74A cells, the most significant alterations after irradiation were linked to tryptophan metabolism. In the more radioresistant UM-SCC-74B cells, the major alterations after irradiation were connected to nicotinic acid and nicotinamide metabolism, purine metabolism, the methionine cycle as well as the serine, and glycine metabolism. The data suggest that the more radioresistant cell line UM-SCC-74B altered the metabolism to control redox-status, manage DNA-repair, and change DNA methylation after irradiation. This provides new insights on the mechanisms of radiation response, which may aid future identification of biomarkers associated with radioresistance of cancer cells. Every year more than half a million new cases of squamous cell carcinoma of the head and neck (HNSCC) are reported , which mRadiotherapy (RT), the clinical application of ionizing radiation, is one of the most effective tools in therapy of cancer today . Even thThe metabolism of cancer cells differs from normal differentiated cells . Tumor pCancer metabolism is altered by ionizing radiation. Radiation exposure induces different types of genome damage, including DNA single- and double-strand breaks and bulky lesions. Thereby multiple signaling pathways are activated, involved in e.g., DNA damage response, signal transduction pathways, and regulation of survival , 12, 13.Several studies have identified different molecular entities associated with radioresistance, nevertheless the underlying mechanisms are still inconclusive . SuggestMetabolic profiling is the comprehensive and quantitative analysis of small endogenous metabolites that are the downstream products in biological systems. It can be a powerful approach to study the phenotype of cancer cells, since it analyses the biochemical outcome of the activities of the proteome . The proEven though our current understanding of the mechanisms in play during radiation of tumor cells, and how they are related to radiosensitivity, is incomplete , it has Consequently, metabolomics may provide insights into the mechanism behind a reduced sensitivity to radiotherapy by identifying differences in metabolites produced in response of irradiation in cancer cells with different sensitivity to radiotherapy. This may provide significant mechanistic understanding related to cellular response due to perturbations caused by radiation treatment , which mIn the present study, we investigate the relationship between radiation response and the metabolome of HNSCC in a unique model system, using two HNSCC cell lines from the same donor but with different radiosensitivity. This enables the study of their metabolic response to radiation in an exceptionally controlled setting, and has to our knowledge not been performed previously. The aim was to assess the metabolic differences between the two cell lines, and how this was affected by radiation. Cells were first genotyped, and assessed for radiosensitivity using clonogenic survival and long-term growth inhibition assays at several radiation doses. Cell-based metabolic profiling using liquid chromatography hyphenated to high resolution mass spectrometry (LC-HRMS) was then performed to investigate the influence of early and intermediate radiation responses on metabolites, and to assess the potential correlation to the different radiosensitivities of the cells. In the long-term, the study may contribute to provide new insights on the mechanisms of radiation response, discover possible biomarkers for radiation-sensitivity, and possibly present potential treatment targets in order to enhance radiation-sensitivity of HNSCC.This study has been conducted in accordance with Frontiers guidelines on study ethics. It does not involve any animal or human subjects or identifiable human data, thus does not require ethical permission.2 in DMEM medium containing 2 mM l-glutamine , supplemented with 5% fetal bovine serum , MEM non-essential amino acids , and antibiotics (100 IU penicillin and 100 μg/ml streptomycin) .The squamous cell carcinoma cell lines UM-SCC-74A and UM-SCC-74B were kindly provided by Professor TE Carey , and have previously been described by Brenner et al. . The celUM-SCC-74A and UM-SCC-74B cells were Genotyped using Short Tandem Repeat (STR) Analysis in order to verify the origin and similarity of the cell lines. DNA was extracted from frozen cell cultures and analyzed using the AmpFLSTR® Identifiler® PCR Amplification Kit. The Identifiler kit amplifies 15 loci and Amelogenin in a single tube and provides loci consistent with major worldwide STR databasing standards.2 culture flasks with 8 ml complete medium. After 48 h, cells were exposed to external beam radiation using 137Cs gamma-ray photons at a dose-rate of ~1 Gy/min , corresponding to a dose of 0, 2, 4, 6, or 8 Gy. Colonies were allowed to form for 10–14 days. Cells were then fixated with 95% ethanol and stained with crystal violet. The colonies were inspected under a microscope, and only cells giving rise to colonies consisting of 50 or more cells were considered clonogenic survivors. Plating efficiency, PE (number of colonies formed/number of cells seeded in the control), and the survival fraction (number of colonies formed after treatment/number of cells seeded × PE) were calculated in Microsoft Office Excel 2016 for Mac version 14.6.1 . Statistical analyses were performed using GraphPad Prism 6 . Differences in normalized survival fractions of 2 Gy irradiated UM-SCC-74A cells vs. UM-SCC-74B cells were assessed using an unpaired t-test and were considered statistically significant if P < 0.05.Clonogenic survival assays were performed as described previously , in orde2 culture flasks with complete medium. After 48 h, cells were exposed to external beam radiation corresponding to a dose of 0, 2, 4, 6, or 8 Gy. Cells were then counted and reseeded about once a week, and the corresponding total cell numbers were calculated. The increase in cell number was followed for 4 weeks. Cell doubling times were calculated using the least square fitting method. In order to determine any statistically significant differences from the untreated group at the last data point, total cell numbers were analyzed using one-way ANOVA followed by Dunnett's multiple comparisons test in GraphPad Prism and were considered statistically significant if P < 0.05.As a complement to the clonogenic- and 3D cell culture assays, the long-term growth inhibitory effects of radiation were evaluated using a growth inhibition assay as described earlier . In shor137Cs gamma-ray photons as described above. Spheroid images were obtained at start of treatment and 10 days after treatment using a Canon EOS 700D camera mounted on an inverted Nikon Diaphot-TMD microscope. The images were analyzed using ImageJ version 1.48 , by measuring the surface area of each spheroid and calculating the volume, assuming each spheroid retained a spherical form. Each spheroid was normalized to its own starting volume at the start of treatment . Statistical analyses were performed using GraphPad Prism 6 . Differences in normalized spheroid growth ratios of UM-SCC-74A cells vs. UM-SCC-74B cells were assessed using an unpaired t-test and were considered statistically significant if P < 0.05.For liquid overlay 3D multicellular tumor spheroid formation, 96-well plates were coated with 0.15% agarose dissolved in PBS with 1% penicillin/streptomycin. One thousand UM-SCC-74B cells/well or 1500 UM-SCC-74A cells/well were seeded and incubated at 37°C in supplemented media for 3 days prior to irradiation with 2 Gy or mock radiation (0 Gy) using 137Cs gamma-ray photons as described above. Whole-cell lysates were prepared 12 h after irradiation according to standard protocols. Cell lysates were diluted 1:1,000. The Cleaved PARP1 Human SimpleStep ELISA® Kit  was used according to the manufacturer's protocol. The OD was then measured at 450 nm using a microtiter plate reader . Statistical analyses were performed using GraphPad Prism 6.Levels of cleaved PARP1 in cell lines were measured using ELISA. The assay detects the presence of the 89 kDa PARP1 fragment containing the catalytic domain. The proteolysis of PARP1 by activated caspase-3 renders the enzyme inactive, which further facilitates apoptotic cell death. Thus, the presence of the 89 kDa PARP1 fragment is considered to be a reliable biomarker of apoptosis. Cells were incubated for 48 h prior to irradiation (2 Gy) or mock radiation (0 Gy) using t-test and were considered statistically significant if P < 0.05.Differences in cleaved PARP1 levels were assessed using an unpaired 6 UM-SCC-74A or (10–25) × 106 UM-SCC-74B cells were cultured in cell culture plates  at 37°C, in 5% CO2 in supplemented DMEM medium. At the time of irradiation, cells were exposed to external beam radiation corresponding to a dose of 0 or 2 Gy. Cells were subsequently harvested at ~75% confluence at 4 h  and 24 h  after irradiation as according to Engskog et al. (2 followed by thawing at 37°C for 10 min. The freeze/thaw cycle was then repeated twice with subsequent sonication on ice for 30 s. Samples were stored at −80°C until metabolite extraction.Two days before irradiation (18–25) × 10g et al. . The timg et al. , 41, 43.2(g).The samples were stored at −80°C after evaporation. Prior to analysis the samples were reconstituted in acetonitrile:Milli-Q water 76:24.Prior to extraction of the intracellular metabolites, the samples were randomized into five separate sample batches comprised of 20 samples each. Each sample batch was prepared and analyzed separately. The samples were thawed at room temperature and centrifuged at 2000 RCF for 10 min at 4°C. A quality control (QC) sample was created for each batch by pooling an equal volume from all samples within each batch. The QC sample was extracted as described below. The five QC samples were pooled after extraction of all five batches. The aqueous supernatants were transferred to fresh extraction tubes followed by addition of chloroform and methanol for the final proportion 2.85:4:4 water:methanol:chloroform –53. The E-mode within the scan-range m/z 50–800. All samples were analyzed in negative ionization mode first, followed by positive ionization mode. The capillary voltage was 1 and −2 kV and the cone voltage was set to 30 and 25 V in positive and negative ionization mode, respectively. In both ionization modes the source temperature was 120°C. Nitrogen was used as desolvation gas at the flow-rate 800 l/h and the desolvation temperature was 500°C and 450°C in positive and negative mode, respectively. Nitrogen was used as cone gas as well at a flow-rate of 50 l/h. and. A collision energy ramp from 20 to 45 eV was used for MSE acquisition with argon as collision gas. Lock-mass correction for accurate m/z measurements was applied using a solution of leucine-enkephalin (m/z 556.2766). Each sample batch was analyzed separately, i.e., one sample batch per day. Prior to each sample batch analysis the instrument was mass calibrated and the sample cone was cleaned. A reference mix  was analyzed before and after each batch to check the system suitability with regard to mass accuracy, instrument sensitivity, and column performance. The QC sample was analyzed repeatedly prior to the study samples for system conditioning, to ensure stable analytical conditions, as well as, between the randomized study samples in regular intervals to monitor the analytical stability throughout the analysis  hyphenated to a G2S Synapt Q-TOF equipped with an electrospray ionization (ESI) source (Waters). All systems were controlled using Masslynx version 4.1 (Waters). For chromatographic sample separation prior to detection a Acquity BEH amide column  from Waters was used. The column temperature was kept at 40°C for all analyses and the injection volume was 5 μl. Mobile phase A consisted of 95:5 acetonitrile/water with 10 mM ammonium formate and 0.1% FA and mobile phase B consisted of 50:50 acetonitrile/water with 10 mM ammonium formate and 0.1% FA. A non-linear elution gradient from 100% A to 100% B was used, the flow rate was set to 0.3 ml/min. In detail: 100% A was kept for 0.5 min then decreased non-linearly (slope-factor 8 in MassLynx) over 12.5 min to 100% B, 100% B was held for 4 min followed by 6 min at 100% A to re-equilibrate the column for a total run-time of 23 min. Detection was performed in both positive and negative ionization mode in resolution MSanalysis .Formic acid, FA(LC-MS grade), ammonium formate (LC-MS grade), methanol (LC-MS grade), Cytidine (99%), hypoxanthine (≥99%), and tryptophan (≥99.5%) were purchased from Sigma Aldrich . Phenylalanine (>99%) was purchased from MERCK  while glutamine (>99%) was purchased from Fluka . Acetonitrile (LC-MS grade) was obtained from Fischer Scientific  and chloroform  was purchased from BDH Laboratory Supplies . Leucine-encephalin was prepared and certified by ERA . The water was purified using a Milli-Q™ water system from MilliPore .Data quality was assessed through in-depth examinations of five representative metabolites spread out in the obtained chromatograms; hypoxanthine, cytidine, phenylalanine, tryptophan, and glutamine. The evaluation was performed by univariate data analysis based on all QC sample injections, in total 25 injections , prior to data pre-processing and multivariate data analysis. Mass accuracy, retention time, and peak area was monitored to check system stability throughout the analysis. DataBridge  was used to convert the raw data files to NetCDF files. The R-based software XCMS was used for peak detection, retention time alignment, and peak grouping . The cenThe reduced and filtered data sets from the data processing were analyzed by multivariate data analysis using SIMCA-P+ . All data was pareto scaled prior to multivariate data analysis. Principal Component Analysis (PCA) was used to find sample clustering, identify possible sample outliers, and systematic trends in the data. Orthogonal Projection to Latent Structures- Discriminant Analysis (OPLS-DA) in combination with S-Plots were used analyse differences between sample groups and to identify differentiating features between sample groups , 61.Comparisons were made between the two cell line controls as well as between irradiated cells and controls of the respective SCC cell lines. The irradiated cells were further divided into subgroups of rapid response and intermediate response depending on the time between cell irradiation and cell sample harvesting. Rapid response subgroups were harvested 4 h after irradiation and intermediate response subgroups were harvested 24 h after irradiation.p-corr values >0.4 were selected and annotated. Molecular weight, isotopic patterns, fragmentation and, when possible, retention time comparison to an in-house database were utilized for feature annotation. The Human Metabolome Database (HMDB), METLIN and in-house databases was utilized to search for the experimental m/z values with a molecular weight difference tolerance of 30 ppm. The raw data signal isotopic pattern, fragmentation (when reference spectra was available) as well as related adducts present at the same retention time in the raw data were all matched against the plausible hits from the data base search. All annotated metabolites should be considered putatively annotated (level 2) according to the Metabolomics Standards Initiative nomenclature  was used for univariate significance testing of all annotated features and p-values <0.05 was considered significant. The significantly altered metabolite are presented as fold changes with 95% confidence intervals. The confidence intervals of fold changes were calculated using Fieller's theorem.Features with nclature . All annSTR results demonstrated identical results for unirradiated UM-SCC-74A and UM-SCC-74B , verifyiThe effect of radiation on UM-SCC-74A and UM-SCC-74B cell viability was studied using clonogenic survival assays, 3D cell growth assays, and long term growth inhibition assays, and can be seen in In the clonogenic survival assay , UM-SCC-in vivo-like environment  in the raw data while glutamine showed huge variations of up to 128%.Multivariate data analysis was performed on the pre-processed and filtered data using PCA and OPLS-DA. All samples were analyzed with the unsupervised model PCA to examine possible sample group separations and sample clustering behavior. The PCA scores plot revealed clear discrimination between the intracellular metabolome of the SCC cell lines in the second component (PC2) . There wThe supervised model OPLS-DA was used to analyse the metabolic changes due to irradiation in the two SCC cell lines as well as the metabolic differences between the SCC cell line controls (unirradiated controls). The metabolic changes after irradiation in each SCC cell line was separated into rapid response (cells were harvested 4 h after irradiation) and intermediate response (cells were harvested 24 h after irradiation), respectively.p < 0.05) metabolic differences between the two non-irradiated cell line controls (p = 0.005). Moreover, guanosine, inosine, xanthine, and the purine intermediate 5-phosphoribosylamine were found at significantly different levels in the two cell lines indicating changes in purine metabolism and biosynthesis between UM-SCC-74A and UM-SCC-74B (p = 0.006).The metabolic profiling demonstrated a number of significant . In contrast, the less radiosensitive cell line UM-SCC-74B showed numerous metabolic alterations after irradiation, where the main metabolic alterations were connected to nicotinate and nicotinamide metabolism (p = 0.003), the methionine cycle (p = 0.05), and purine metabolism (p = 0.001)  . A numbein vitro model, in which two HNSCC cell lines from the same donor were employed. These cells exhibited the same STR genetic profile  was found increased 24 h after irradiation the UM-SCC-74A cell line, which might suggest an increase in serotonin degradation to 5-HIIA. Serotonin has been shown to exhibit growth stimulatory effects on several types of carcinoma and other tumor cells, and to play a role in radiation-induced bystander effect . In prevIn the UM-SCC-74A cell line, no investigated metabolites in the nicotinamide and nicotinic acid metabolism demonstrated significantly altered levels after irradiation. This is in contrast to the UM-SCC-74B cell line, where the metabolites nicotinamide, 1-methylnicotinamide, nicotinamide ribotide, nicotinic acid mononucleotide, NADH as well as NAD+ were all almost depleted 24 h after irradiation . This dein vitro from oxidative damage induced by reactive oxygen species (ROS), and to serve as a radioprotector  and guanosine monophosphate (GMP) were decreased (not significantly) 24 h after irradiation. This indicates a functioning purine salvage pathway with an increased degradation of the purine nucleotides to the corresponding nucleosides and nucleobases. This is in line with previous studies, where guanosine have been shown to protect DNA rotector . Adenosirotector . In contThe pathway of cysteine and methionine metabolism was altered in the UM-SCC-74B cell line after irradiation, in contrast to UM-SCC-74A cells . S-adenoOur data strongly implicates that the radioresistant cells changed their metabolism to control the redox status, DNA repair as well as DNA methylation. A lot of preclinical efforts have over time been devoted to the development of strategies to sensitize cancer cells to radiation therapy. This study was a first step in the understanding of which metabolic pathways in SCC that were important for the differences in radiosensitivity between the two cell lines UM-SCC-74A and UM-SCC-74B. The elucidation of the mechanisms behind radioresistance could lead to better prediction of radiation treatment outcome or possibilities to sensitize tumors to radiation. However, all metabolites and metabolic pathways investigated in this study all require further investigation as to whether they will be able to pose as targets for prediction of radiation response or to enhance radiation sensitivity.The datasets for this study are available on request.EL, IE, ME, JH, TA, CP, GL, and MN designed the study. EL, IE, ME, JH, TA, MH, CP, GL, and MN contributed to data analysis and interpretation, and revised the manuscript. EL and IE contributed to experimental studies and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adolescents living in sub-Saharan Africa constitute a vulnerable population at significant risk of HIV infection. This study aims to evaluate the acceptability, feasibility, and accuracy of home-based, supervised HIV self-testing (HIVST) as well as their predictors among adolescents living in Kisangani, Democratic Republic of the Congo (DRC).A cross-sectional, door-to-door survey using a blood-based HIV self-test and a peer-based supervised HIVST approach was conducted from July to August 2018 in Kisangani, DRC. The acceptability and feasibility of HIVST were assessed among adolescents’ consenting to use and interpret HIV self-test. The accuracy of HIVST was estimated by the sensibility and specificity of adolescent-interpreted HIV self-test. Factors associated with acceptability and feasibility of HIVST were analyzed with logistic regression.versus 100%; adjusted OR: 0.01 [95% CI: 0.004–0.03]). In the hands of adolescents at home, the sensitivity of the Exacto Test HIV Self-test was estimated at 100%, while its specificity was 96.0%. The majority of participants (68.0%) affirmed that post-test counseling was essential, and that face-to-face counseling (78.9%) was greatly preferred.A total of 628 adolescents  aged between 15 and 19 years were enrolled. Acceptability of HIVST was high (95.1%); 96.1% of participants correctly used the self-test, and 65.2% asked for verbal instructions. The majority of adolescents (93.5%) correctly interpreted their self-test results. The Cohen’s κ coefficient between the results read by adolescents and by supervisors was 0.62. The correct interpretation decreased significantly when adolescents had no formal education or attended primary school as compared to those currently attending university (37.0% Home-based, supervised HIVST using a blood-based self-test and peer-based approach can be used with a high degree of acceptability and feasibility by adolescents living in Kisangani, DRC. Misinterpretation of test results is challenging to obtaining good feasibility of HIVST among adolescents with poor educational level. Face-to-face post-test counseling seems to be preferred among Kisangani’s adolescents. Adolescents aged between 10 and 19 years . Nearly Despite the need for HIV testing among adolescents, coverage and uptake seemingly remain poor . Thus, lIn the Democratic Republic of the Congo (DRC), the largest French-speaking country in Central Africa, HIV infection is characterized by its heterogeneous prevalence throughout the country estimated to 1.2%, with adolescents constituting 23% of the population but carrying 14% of the burden of the HIV epidemic –12. In 2This study consisted of a cross-sectional, door-to-door survey using a blood-based HIV self-test   and a peCliniques Universitaires de Kisangani, the Hôpital Général de Référence de Kabondo, and the health centers of Neema and Saint Joseph were selected for confirmatory HIV testing, counseling, and care.The study was conducted from July to August 2018 in Kisangani, the capital city of Tshopo province and the third-largest urbanized city in the DRC , comprisThe survey targeted adolescents during the vacation period. All participants volunteered to take part in the study. The inclusion criteria were as follows: ignorance of own HIV status, willingness to be tested for HIV infection, being between 15 and 19 years of age, ability to speak and read the French, Lingala, or Swahili languages, and signed informed consent .Individuals who were already aware of their seropositivity for HIV, who were away from home during the visiting time of the study team, who were unable to communicate in French, Lingala, or Swahili, or who did not provide consent to participate were excluded from the study.n, of households (level 3 cluster), corresponding to n = N/10, was randomly selected, where N was the sample size of the corresponding neighborhood. The number of households to be visited per cluster was determined in proportion to the size of the cluster. Thus, eligible adolescents who were identified at home were enrolled from the selected households at the time of the study visit by the peer educators and counsellors. The population of adolescents in Kisangani was estimated to be 300,000, with a sample size calculated at a 95% confidence level, 0.5 standard deviations, and a 5% confidence interval was estimated at 384 participants.Random sampling in clusters at three levels was used to select survey participants. The first level corresponded to the different neighborhoods in each of the four townships arbitrarily included in the study, using probability proportional to size sampling. After calculating the demographic weight of each commune and determining the size of the corresponding sub-sample, the neighborhoods (level 1 cluster) in each commune were identified and enumerated. In each neighborhood, 10 streets (level 2 cluster) were randomly selected. On each street, the number, In the study, peer educators were adolescents who conducted home-based enrollment of participants to eliminate psychological and social barriers ,18. The The respondent-driven sampling method was used to recruit peer educators. A total of 80 peer educators were originally recruited. After evaluation using a post-training semi-structured questionnaire, considered successful when at least 80% of answers were correct, 36 peer educators were ultimately retained. Note that, the peer educators were also recruited during the vacation period; their participation was voluntary; and only their restoration and transportation costs were taken care of.The main study outcomes were the acceptability and feasibility of HIVST. Acceptability was defined as consenting to and using the provided self-test in the participants’ homes. The feasibility of HIVST was evaluated according to adolescents’ ability to use the finger-stick whole-blood self-test autonomously or with verbal instructionsuntil obtaining a valid test result, and to correctly interpret the HIV self-test result in a home-based, supervised setting.versus voluntary counseling and testing [VCT]), willingness to pay for HIVST, and counseling (pre-test or post-test), as previously mentioned , wh, wh12], The study was conducted after obtaining ethical approval from the Ethics Committee of the School of Public Health of the University of Kinshasa, constituting the National Ethics Committee of the DRC, and from the Ethics Committee of the University of Kisangani. Further permission was obtained from the Tshopo Provincial Health Division. Written informed consent was obtained from all participants or from their parents or guardians if the participants were minors (15 to 17 years) . AnonymiData were entered into an Excel database and analyzed using SPSS 20.0 . Proportions were calculated for all categorical variables. A one-sided Wald asymptotic test was used to assess the confidence interval (CI) of difference in acceptability and preference for HIVST before and after self-testing . HoweverThe sensitivity and specificity of adolescent-interpreted HIV self-test results were calculated, taking into account only the positive and negative test results. Note that, all reactive results confirmed with serologic testing algorithms were considered as a reference for “positive” results. Furthermore, the PPV and NPV were calculated with respect to the reported HIV prevalence in this study using Bayes’ formulae .P-value ≤ 0.05 in the bivariate analysis were entered into the multivariate analysis. Note that, missing data were assigned the null value for conservative estimates.Finally, multivariate regression was used to identify factors independently associated with main study outcomes. Only statistically significant factors at a A total of 353 households were enrolled by the study teams, comprising 679 potential eligible participants. Among them, 628 (83.3%) were successfully enrolled in the study .Baseline characteristics of study adolescents are shown in P < 0.0001). Confidentiality concerns were cited by a majority of participants both before and after HIVST . The rate of acceptability for recommending HIVST to others was higher (96.3%) after HIVST, whereas it was lower (82.5%) before HIVST, giving a difference of -13.8% (P = 0.004); difference was of -10%  concerning the acceptability to further distribute the self-test.Of the 628 adolescents, 597  accepted the test . The reaWhen determining factors associated with the willingness to accept using the self-test via bivariate analyses, no association could be found.versus 55.8%; difference: −30.6%; P < 0.0001). Willingness to pay for the self-test was low in the present study’s series, without a significant difference before versus after performing the test  The majority of participants (68.0%) claimed that post-test counseling was essential after HIVST, whereas only 46.5% claimed this before HIVST ; face-to-face counselling was greatly preferred over other modes of counseling, without a significant difference between the pre-test and post-test phases (78.3% versus 78.9%).Individual preferences for HIVST strategy over conventional testing strategy (rate of substitution of voluntary counseling and testing [VCT] for HIVST), for willingness to pay, and for counseling are presented in Overall, among the 597 adolescents who accepted the use of the HIV self-test, 574  correctly used the self-test and succeeded in obtaining an interpretable result, whereas only 23  failed. The correct use of the HIV self-test was similarly high in girls (95.2%) and boys (97.6%). Easy and correct identification of the components of the HIVST kit was observed in 94.8% (95% CI: 92.7–96.3).versus 57.3%; P = 0.001). Participants requested verbal instructions when interpreting the HIVST results (67.1%) or when using the lancet (42.2%).Almost one-third  of the participants carried out the self-test without any help, whereas 65.2% (95% CI: 61.3–68.9) asked for verbal intructions—girls more so than boys  adolescents correctly interpreted their self-test results, whereas 39  failed to do so. Misinterpretation of self-test results accounted for 30.4% of invalid tests  and 5.8% of negative tests . No positive test was misinterpreted. The Cohen’s κ coefficient between the results read by adolescents and by supervisors was 0.62. The feasibility of HIVST is summarized in versus 100%; P < 0.0001; cOR: 0.01 [95% CI: 0.001–0.02]; aOR: 0.01 [95% CI: 0.001–0.03]). Note that successful performance of HIVST was not associated with any variables in multivariate analysis.Concerning possible factors associated with the feasibility of HIVST, the dependent variables used for this analysis were “successful performance” and “correct interpretation of self-test result.” The following factors were significant in unadjusted bivariate analysis: “age group,” “educational level,” “sexual intercourse in the past six months,” and “knowledge about HIV self-testing’s existence.” Multivariate analysis followed by logistic regression showed that only the variable “educational level” remained associated with the variable “correct interpretation of self-test result” : The corAll 19 adolescents confirmed as being HIV positive with the Exacto Test HIV Self-test (Biosynex) were further confirmed as positive with the national HIV testing algorithm , giving In the present study in Kisangani, DRC, HIVST was highly acceptable (≥ 95%) among adolescents aged between 15 and 19 years. Furthermore, the vast majority (≥ 96%) of participants correctly carried out the self-test. Despite frequent (65.2%) verbal instructions, only 93.5% correctly interpreted the self-test results. Finally, adolescents generally (78.9%) preferred face-to-face counseling and mostly (68.0%) post-test counseling. Taken together, these observations demonstrate that adolescents constitute a target population for HIVST with a high acceptability rate, but they also indicate the intrinsic difficulties encountered in terms of both the interpretation of the results and the demand for counseling about the test.versus finger-stick self-testing does not constitute a barrier against HIVST that should have been addressed prior to implementing HIVST among adolescents.A high acceptability rate for HIVST was observed among study adolescents. High acceptability for the oral fluid-based HIVST were previously reported among adolescents , 25–28, The most important factor influencing the acceptability of HIVST is individual motivation to perform HIV testing . Our stuCommunity-based HIV testing has greatly increased the overall rate of HIV testing in sub-Saharan Africa in the last decade ,15. In tThe sensitivity of adolescent-interpreted HIV self-test results was estimated at 100%, with a specificity at 96.0%. The WHO recommends a success rate higher than 95% on the major items concerning HIVST practicability, including the interpretation of test results . The difSimple and easy-to-use devices, instructions for use, and other support tools have been shown to be key to good performance in sub-Saharan Africa, yet may be highly contextual ,14. The Concerning preferences, the current study showed that the rate of substitution of VCT for HIVST increased after performing the self-test, although the willingness to pay for the self-test was low. In addition, face-to-face post-test counseling was greatly preferred by the participants. Many previous studies in key and general populations have also reported a preference to have counseling available ,37–40.Our study is original because focusing on the vulnerable adolescent population, who is at high-risk for HIV infection and for whom HIV self-testing could constitute a complementary solution for HIV testing access, in the frequently poorly documented cultural context of French-speaking sub-Saharan Africa. The study has however some limitations. First, the factors associated with acceptability of HIVST were not clearly assessed because a small number  of study participants did not accept the HIV self-test. Second, selection bias was likely, as the study population comprised mostly women. Indeed, as the dominant emotional responses, fear and anxiety regarding HIV testing were mainly described in females . FinallyHIVST is a novel means to make testing more accessible, confidential, and available at non-traditional venues, such as pharmacies and community venues, as well as in the home . The preS1 File(XLS)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S7 File(DOCX)Click here for additional data file.S8 File(DOC)Click here for additional data file.S9 File(DOC)Click here for additional data file.S10 File(TIF)Click here for additional data file.S11 File(TIF)Click here for additional data file.